Absorption and fluorescence spectra
The absorption spectrum of EG without DNA presence showed a major peak at 470 nm and a shoulder peak at around 495 nm (Figure 1). Upon addition of increasing amounts of dsDNA (λDNA), the peak at 470 nm dropped while a new peak at 500 nm emerged. At saturating DNA concentrations, the 470 nm peak decreased to a shoulder peak while the peak at 500 nm became the major peak. Without DNA present, the dye was weakly fluorescent with an excitation peak at 495 nm and emission peak at 525 nm (Figures 2). In the presence of dsDNA, the excitation and emission wavelengths of the dye red shifted to 500 nm and 530 nm, respectively. By comparing the dye's fluorescence intensities at zero and saturating (i.e., 40 ng/μL) λDNA concentrations, the fluorescence enhancement of the dye upon DNA binding was estimated to be approximately 70 times (Figure 2). In the presence of the same concentration of M13 DNA, which is considered to be mostly single-stranded with some local double-stranded regions, the fluorescence enhancement was only about 21 times.
DNA titration
To gain further insight into the fluorescence responses of the dye toward dsDNA and ssDNA over a wider range of DNA concentrations, the dye was titrated with λDNA and M13 DNA, respectively, in 1× AmpliTaq buffer. The fluorescence intensity readings for each titration were plotted against the DNA concentrations as shown in Figure 3. The linear titration range for λDNA was from 0 to about 10 ng/μL with a slope of 0.148 while the linear titration range for M13 DNA was from 0 to at least 40 ng/μL with a slope of 0.013. The ratio of the two slopes is thus 11.4, indicating that within the linear titration range the dye is more than 11 times more fluorescent in the presence of dsDNA than in the presence of ssDNA of the same weight concentration.
In order to assess whether EG binds to dsDNA with any base preference and whether its DNA binding is affected by salts, the dye was titrated with the double-stranded AT-rich λDNA and the double-stranded GC-rich Tbr DNA, respectively, in Tris buffers with or without an inorganic salt. Furthermore, in order to mimic the condition used during qPCR, the titrations and fluorescence recordings were made at 50°C. As shown in Figure 4A, the fluorescence of the dye in the presence of λDNA (open square) is twice as intense as that in the presence of Tbr DNA (open diamond) in 100 mM Tris pH 8.0. A similar trend was observed when the titrations were carried out in 10 mM Tris pH 8.0 containing 100 mM KCl (Figure 4A, solid square and solid diamond) except that the titration curves were shifted toward the right relative to their counter parts. The large fluorescence difference observed between titration using λDNA and that using Tbr DNA was apparently not due to difference in the dye's binding affinities toward the two types of DNA. This can be better appreciated by replotting the titration curves with normalized fluorescence intensities. As shown in Figure 4B, the normalized titration curves of EG with λDNA and Tbr DNA in 100 mM Tris pH 8.0 are nearly completely superimposable, suggesting that EG binds to the two types of DNA with equal affinity. Likewise, nearly identical normalized titration curves were obtained for EG titrations with λDNA and Tbr DNA in 10 mM Tris pH 8.0 containing 100 mM KCl except that, again, they are shifted to the right relative to the titration curves obtained in 100 mM Tris buffer pH 8.0 (data not shown). The decrease in the dye's DNA binding affinity due to salt effect, as suggested by the shifting of the DNA binding curves to the right, was also observed when the 100 mM KCl in the buffer was replaced with 100 mM MgCl2 (data not shown).
For comparison, SG was also titrated with λDNA and Tbr DNA, respectively, in 100 mM Tris pH 8.0, and the resulting normalized titration curves were co-plotted with those of EG (Figure 4A and 4B). Unlike EG, SG displayed differential binding affinities towards the two types of DNA; the significant left-shifting of the SG titration curve with λDNA suggests that SG has a higher affinity toward λDNA than toward Tbr DNA. Furthermore, as shown in Figure 4B, SG has an overall higher DNA binding affinity than EG does. However, similar to EG, SG also produced nearly 50% less fluorescence when titrated with the GC-rich Tbr DNA, compared to titration with the AT-rich λDNA (data not shown).
To learn how EG and SG each may bind to primers, primer-dimers and primer-template hybrids in typical PCR reactions, each dye was titrated with a 10-base paired stem-loop fragment (ds10-mer), a 22-base paired stem-loop fragment (ds22-mer), and a 24-base single-stranded fragment (ss24-mer). To ensure the conformation stability of the double-stranded fragments and detection sensitivity, the comparative binding study of the dyes with the three DNA fragments was carried out at room temperature instead of a higher temperature as used in PCR. As shown by the normalized DNA binding curves in Figure 5, both EG and SG exhibited lower binding affinity toward shorter dsDNA fragment (i.e., ds10-mer) than longer dsDNA fragment (i.e., ds22-mer), and even lower affinity toward the ssDNA fragment except that EG generally showed lower affinity than SG for each given type of DNA fragment. As the DNA concentration corresponding to the midlevel fluorescence of a binding curve may be used to estimate the relative dissociation constant of a DNA-dye complex [24], it is evident that the degree of binding affinity decrease for EG as a result of DNA fragment size reduction or change from dsDNA to ssDNA fragment was far more pronounced than that for SG.
Effect of dye concentration on qPCR performance
SYBR Green I is known to show PCR inhibition when used at above a certain threshold concentration [9, 11, 25, 26]. The exact threshold concentration may vary, depending on such factors as the type or make of DNA polymerase, primer sequences, amplicon, buffer components and cycling parameters. It is desirable to have a relatively high threshold dye concentration so that PCR signals can be made more robust and DNA melt curve data more reliable (See discussion section). To explore the concentration limit for EG, we conducted qPCR experiments using four separate dye concentrations, 2.66 μM (2×), 2 μM (1.5×), 1.33 μM (1×) and 0.67 μM (0.5×), respectively. Also, to test the effect of amplicon size, each dye concentration was applied to the amplifications of three separate amplicons, McG (121 bp), TBP (228 bp), and GCL (529 bp), all cloned into a plasmid (Figure 6, Panels A1, B1 and C1). For comparison, similar experiments were carried out using four SG concentrations, 1.36 μM (2×), 1.02 μM (1.5×), 0.68 μM (1×) and 0.34 μM (0.5×), respectively, under the same condition (Figure 6, Panels A2, B2 and C2). The concentrations of SG were determined using a SG extinction coefficient of 58,000 at 494 nm, which we estimated using a combination of the dye's optical density specification supplied by the manufacturer and published information on the dye's structure [27]. This number is significantly lower than the reported extinction coefficient of 73,000 (see detailed explanation in Methods).
To eliminate any differences due to primer annealing, the universal M13 forward and reverse primers were used for all PCR experiments. Also, the denaturing and annealing steps in each thermo cycle were each set for 15 seconds to ensure that the two processes were as close to completion as possible in all experiments.
Using the standard PCR protocol (60 seconds at 72°C), EG at 0.5× (i.e., 0.67 μM) and 1× both gave relatively early Ct for all three amplicons. Slight Ct delays occurred at 1.5 × EG while raising the dye concentration to 2× caused significant Ct delays (Figure 6, Panels A1, B1, and C1). For SG, the only non-inhibitory concentration was 0.5× (i.e., 0.34 μM) for all three amplicons (Figure 6, Panels A2, B2 and C2). Higher SG concentrations resulted in either significant Ct delay or non-specific amplifications as revealed by melt curve analysis and gel electrophoresis (data not shown). Thus, based on these results, the threshold concentrations (i.e., the highest possible dye concentrations without causing significant Ct delay and nonspecific product formation) under our conditions are approximately 1.33 μM for EG and 0.34 μM for SG, respectively. As shown in Figure 6, qPCR using EG on average gave 4–5 times more fluorescence than that using SG when both dyes were applied at their threshold concentrations. It is also worth noting that the effect of target sequence on Ct differed slightly between reactions using the two dyes. For example, for the amplifications of McG and GCL, EG gave earlier Ct values than SG. On the other hand, for the amplification of TBP, SG gave earlier Ct values than EG. In general, however, the Ct difference between two dyes for a given amplicon was within ± 1 cycle.
Effect of chain extension time and amplicon size on qPCR performance
Given the finding that EG had relatively low PCR inhibition, we reasoned that it might be possible to reduce the chain extension time from the standard 60 seconds to a shorter time without adversely affecting PCR performance. Thus, we compared EG and SG in qPCR experiments using the extension time at 72°C of 30, 15 and 5 seconds, respectively. In order to see the interplay of dye concentration and chain extension time, each dye was also tested at two different concentrations, i.e., 0.67 μM (0.5×) and 1.33 μM (1×) for EG and 0.34 μM (0.5×) and 0.68 μM (1×) for SG, respectively. Again, McG, TBP and GCL were chosen to represent amplicons of different sizes in the comparative studies.
As shown in Figure 7, panel A1, reduction of chain extension time did not significantly affect the Ct values for McG amplifications for either dye at 0.5× concentration although amplifications with EG consistently gave about twice the final fluorescence signal as those with SG did. Doubling the concentration of EG from 0.5× to 1× had no major effect on the amplification in terms of both Ct values and specificity (Figure 7, panel A2). In contrast, when the concentration of SG was doubled to 1×, the Ct became increasingly delayed as the chain extension time was systematically shortened. When the extension time was shortened to 15 seconds or lower, there were marked Ct delays as well as formation of nonspecifically amplified products (Figure 7, panel A2, lines S7 and S8) as confirmed by post-run DNA melt curve analysis (data not shown).
Similar results were obtained in the comparison of EG and SG in the amplifications of the longer 228 base pair TBP and 529 base pair GCL, respectively, except that the Ct delay for the amplifications using SG became even more pronounced as the chain extension time was shortened. Amplification reactions using EG showed significant Ct delay, only under the most challenging condition–the combination of a very long amplicon (529 bp), a relatively high dye concentration (1× or 1.33 μM) and a very short extension time (5 s) (Figure 7, panel C2, line E8). On the other hand, the Ct values for amplification reactions using SG became quite sensitive to reduced extension time, especially at the higher 1× (0.68 μM) dye concentration (Figure 7, panels B2 and C2). Moreover, when the extension time was shortened to 30 seconds or below, amplifications of both TBP and GCL using SG produced nonspecific products (Figure 7, panels B2 and C2, lines S7 and S8). In fact, when the extension time was reduced to 5 seconds, only nonspecifically amplified products were detected as revealed by post-PCR DNA melt curve analysis and gel electrophoresis (data not shown). Finally, reactions using SG generally showed poorer reproducibility than those using EG in response to decreasing extension time and elevated dye concentration (data not shown).
The above results suggest that EG may be suitable for fast cycling qPCR. Thus, to further explore this possibility, we carried out qPCR on a GAPDH fragment using the optimal 1.33 μM EG concentration. The reactions were performed using two-temperature cycling protocols of 15 seconds at 95°C and various amounts of reduced annealing/extension time (i.e., 60, 40, 20 and 10 seconds, respectively) at 60°C. For comparison, a commercially available kit, Power SYBR Master Mix, was run side-by-side, also using the same two-temperature cycling protocols. The commercial master mix contained approximately ~0.3 μM SG as determined from the dye's optical density at 494 nm (See Method section for SG concentration determination). Instead of making our own SG master mix from the same buffer and enzyme system used for EG, we chose Power SYBR Master Mix to avoid the possibility that the our buffer and enzyme system might show bias against SG and because Power SYBR Master Mix was of optimized formulation for SG as suggested by the manufacturer. Each amplification reaction was repeated eight times in order to assess the reproducibility of the reaction. Figure 8 displays the Ct values of amplification reactions run with various reduced annealing/extension times for Power SYBR Master Mix and the EG master mix. When the annealing/extension time was set for 60 seconds, amplifications with either master mix gave comparable Ct values and similar reproducibility. However, as the annealing/extension time was shortened, the Ct for Power SYBR Master Mix became increasingly delayed and its reproducibility, as shown by the error bars in the figure, also suffered progressively. For example, the Ct for Power SYBR was delayed by as many as 5–6 cycles when the annealing/extension time was reduced to 10 s. On the other hand, for the same extent of annealing/extension time reduction, the Ct for the EG master mix increased by only about 1 cycle while still maintaining excellent reproducibility.
PCR dynamic range
A key application of qPCR is to determine the starting concentration of a target gene. This is typically accomplished by comparing the Ct for the target gene amplification of the sample against a standard qPCR titration curve, where the Ct is linearly related to the logarithm of the starting DNA copy number. Among other factors, the linear dynamic range of the PCR titration is expected to be affected by the qPCR dye. Thus, to compare EG and SG for their performance in this aspect of qPCR, 10-fold serial dilutions of a GAPDH target as a cDNA preparation were amplified using either an EG master mix or Power SYBR master mix. Following each PCR experiment, DNA melt curve analysis was carried out to verify amplification specificity. As shown in Figure 9, target amplifications in the presence of either dye produced comparably good linear dynamic range, efficiency and specificity. However, amplifications using the EG mix resulted in significantly earlier Ct values and higher signals than those using Power SYBR mix. In addition, the fluorescence signals continued to rise for more than 30 cycles for amplifications using the EG mix. On the other hand, for amplifications using Power SYBR mix, the signal increase lasted for only about 10 cycles before reaching a plateau.
DNA melt curve analysis
We observed that DNA melt peaks associated with EG at its optimal concentration (i.e., 1.33 μM) were significantly stronger and also narrower than those associated with SG at its optimal concentration. In addition, the melt temperatures for DNA associated with EG were generally from 0.5 to about 1°C lower than those for DNA associated with SG in the same buffer. Figure 10 compares the melt curves of two GAPDH amplicons, each resulting from the amplification of 100 pg starting human cDNA using either a EG mix or Power SYBR mix as described above for the human cDNA titration experiments. As shown in the figure, the peak width at half height for EG (~1.2°C) is only about half of that for SG (~2.2°C) while the peak height for the former is nearly ten times that for the latter. In order to rule out the possibility that the apparently wider peak associated with SG was due to multiple peaks superimposed with each other, we examined the products from both PCR reactions on agarose gels. The results indicated that the PCR products from both reactions were the same single product.
Effect of DNA polymerase source on the comparative performance of EG and SG in qPCR
The qPCR data shown in Figure 6 and 7 were all generated with AmpliTaq from ABI. As DNA polymerases from different commercial sources may be prepared differently and supplied in different storage buffers, we would like to find out if the make of enzyme played any role in the observed qPCR performance difference between EG and SG. The experiments were repeated with Taq from NEB, Promega, Fermenta and home made Taq respectively. We found that although Ct values and PCR signal strength differed slightly from enzyme to enzyme and from run to run, the performance difference between the two dyes still held (data not shown).
Stability of EvaGreen
qPCR exposes reaction components to high temperature and, in some PCR instruments, to high powered laser irradiation. Moreover, the pH of Tris-based master mixes is often alkaline at room temperature and especially at 4°C during storage. Thus, it is essential for a qPCR dye to be thermally, hydrolytically and photolytically stable. To test the thermal and hydrolytical stability of EG under an accelerated condition, a solution of the dye along with a reference dye ROX was incubated in pH 8.0 Tris at 99°C for three hours. During the course of incubation, the solution was periodically sampled for absorption spectral measurement at room temperature. For comparison purpose, a solution of SG and ROX reference dye in the same buffer was incubated and monitored under the same condition. As shown in Figure 11, the absorption spectrum of EG shifted only slightly, a result largely within the experimental error. In contrast, the spectrum of SG had nearly disappeared at the end of the 3-hour incubation. Similar data were obtained when the dyes were incubated in pH 9.0 Tris buffer at the same temperature except that SG decomposed even faster (data not shown). To test the ability of EG to withstand multiple freeze-and-thaw cycles, a 26.7 μM EG solution in H2O (EvaGreen 20× from Biotium, Inc.) was subject to 10 freeze-and-thaw cycles by taking the sample in and out of a -20°C freezer over the course of one week. UV/Vis measurements of the EG sample before and after the freeze-thaw test detected no significant change (data not shown). EG also appeared to be reasonably photostable as no signal variation was observed during routine qPCR experiments due to photobleaching of the dye.